Within the United States, an estimated 2.1 million people suffer from addiction to opioids, considered the most lethal drugs of abuse. Current research has made significant progress with regard to the cellular and molecular mechanisms of drugs of abuse and identified the neural substrates that are affected by addiction. To develop improved therapeutic treatments, however, requires a deeper understanding of how opioids compromise brain plasticity. To examine how opioids affect brain plasticity, the project uses zebrafish in conjunction with powerful volumetric (3D) imaging approaches including fluorescence-based Light Sheet Microscopy (LSM) and Magnetic Resonance Imaging (MRI) technologies as well as molecular profiling and gene expression analyses. The overarching goal of the study is to develop a network model of those neural systems that control reward-seeking and motivated behaviors in zebrafish. To dissect these circuits and understand how opiods affect the plasticity of the brain, the two aims of the project combine powerful imaging technologies with behavioral experiments, neural activity readouts, tract tracing, and molecular and gene expression analyses in zebrafish. Aim 1 integrates behavioral experiments with immunohistological detection of phosphorylated ribosomal protein (p)S6 as a readout to map functional circuitry during associative reward-seeking and addiction behavior in adult zebrafish. Molecular profiling (phosphor-trap) of activated neurons and RNAseq experiments will reveal quantitative and qualitative gene expression changes during addicted states. Aim 2 combines several magnetic resonance imaging methodologies to image whole brain circuitry and neurochemical characteristics. Histological data derived from both LSM and epi-fluorescence microscopy will be integrated to generate a 3D-segemented MRI atlas that can be used in functional MRI studies employing live zebrafish. The results of the project will lead to a comprehensive network model of reward and decision making circuitry critical for using zebrafish as a model for addiction. Ultimately, the project will identify how opioid use affect brain plasticity across levels of organization.